Abstract

Neuronal synchronization in the olfactory bulb has been
proposed to arise from a diffuse action of glutamate released from
mitral cells (MC, olfactory bulb relay neurons). According to this
hypothesis, glutamate spills over from dendrodendritic synapses formed
between MC and granule cells (GC, olfactory bulb interneurons) to
activate neighboring MC. The excitation of MC is balanced by a strong
inhibition from GC. Here we show that MC excitation is caused by
glutamate released from bulbar interneurons located in the GC layer.
These reciprocal synapses depend on an unusual,
2-amino-5-phosphonovaleric acid-resistant,
N-methyl-d-aspartate receptor. This type of
feedback excitation onto relay neurons may strengthen the original
sensory input signal and further extend the function of the dendritic
microcircuit within the main olfactory bulb.

The first relay in olfactory
information processing is the main olfactory bulb where synaptic
transmission between dendrites represents the major mechanism for
neuronal interaction (1–5). At this level, synaptic transmission
includes both inhibitory and excitatory signals that coexist in a
purposeful balance. Inhibition is provided by a reciprocal
dendrodendritic circuit that forms the basis for a reliable, spatially
localized, recurrent inhibition of mitral cells (MC). Hence, glutamate
released by lateral dendrites of bulbar relay neurons, MC, and tufted
cells excites the dendrites of local interneurons called granule cells
(GC) (6–8), which in turn, release γ-aminobutyric acid (GABA)
directly back onto MC (7–10). The extensive lateral dendrites of MC
and the possible spread of excitation through GC dendrites provide a
mechanism for lateral inhibition (7, 8, 11–13). Finally, because a
single GC is believed to contact a large number of MC (14), this
reciprocal inhibitory synaptic connection contributes to the
synchronization of MC (15–17). As a result, feedback inhibition has
been proposed to be crucial for the complex dynamics of olfactory
network responses (18).

In addition to these inhibitory inputs arising from GC, it has been
reported that MC lateral dendrites receive large excitatory inputs when
either inhibition was antagonized or magnesium was removed from the
external medium (19–22). In mammals, excitatory synapses onto MC have
been localized exclusively to the apical dendritic tufts that receive
primary sensory afferents (2, 4, 5, 23, 24). The origin of the
excitatory inputs to the MC lateral dendrites therefore is debated.
Isaacson (20) has proposed that this excitation depends on glutamate
release from the MC themselves. However, it is unknown how the
specificity for odor processing can be conserved if glutamate spillover
alone governs excitatory transmission within the main olfactory bulb.
Using a combination of in vitro whole-cell recordings and
immunogold detection of glutamate, we explored the possibility that
ionotropic glutamate receptors on MC could rather be activated by
interneurons located in the GC layer. Such feedback excitation would
provide an effective mechanism for temporal and spatial codings in
olfactory information processing.

l-glutamate (250 mM), prepared in distilled
H2O and adjusted to pH 8.2 with NaOH, was
successively applied iontophoretically to the MC lateral dendrite or
soma. No pharmacological differences were observed between these two
locations (20- to 300-nA current application during 500 ms and 15 nA
for continuous retention) through a 5–10 MΩ patch pipette by using a
dual microiontophoresis current generator (World Precision Instruments,
Astonbury, U.K.). Applications were performed every 20 s. All
evoked currents were subtracted with the traces recorded in the
Mg2+-free external medium containing: 0.5 μM
tetrodotoxin (TTX), 10 μM bicuculline methiodide (BMI), 100 μM
picrotoxin (PTX), 10 μM
1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide
(NBQX), 100 μM
d,l-2-amino-5-phosphonovaleric
acid (APV), and 15 μM 5,7-dichlorokynurenic acid (dCK).
l-glutamate also was applied iontophoretically (30- to
40-nA current application during 2.5 s and 10 nA for continuous
retention) to the GC soma (stimulations were performed every 20
s). The electrode was placed in the GC layer at least 100 μm away
from the MC layer to prevent any direct effect of glutamate on MC.
Stimulation of GC by iontophoresis of glutamate was chosen because it
prevents MC antidromic spike generation or centrifugal fibers
stimulation. Glutamate application elicited by low current
injection/long duration was the best way to prevent direct effect on
MC and to promote spike generation by glutamate receptor activation. As
control, we have performed GC current-clamp recordings to check that
iontophoresis of glutamate was efficient in eliciting spikes even in
the presence of APV and dCK (data not shown).

Synaptic responses were filtered at 1–5 kHz with an eight-pole Bessel
filter, digitized at 4 kHz on a TL-1 interface (Axon Instruments,
Foster City, CA), and collected on an IBM-compatible computer. On- and
off-line data analyses were carried out with
acquis-1 (Gérard Sadoc, Centre
National de la Recherche Scientifique). During all experiments, the
access resistance (Ra) and the input
membrane resistance (Rm) were
monitored, and acquisition was terminated when these parameters changed
more than 20%. Data are expressed as mean ± SEM, and for
statistical comparisons we used a paired Student's t test.

When Lucifer yellow (0.4%) was added in the recording pipette no
difference in the frequency of small excitatory postsynaptic currents
(EPSCs) was observed between MC having their apical dendrite sectioned
or not. However, the large spontaneous inward currents previously
described were eliminated in apical dendrite-free MC (20–22, 25).
Furthermore, kinetic analysis allows us to discriminate between EPSCs
originating from the olfactory nerve and those from lateral
dendrites. Drug solutions were bath-applied by using a
gravity-driven perfusion system. NBQX,
d,l-APV,
3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP), MK-801,
dCK, d-serine, AP7, and LY 235 959 were provided from
Tocris; all other drugs and salts were purchased from Sigma.

Electron Microscopy Data.

Postembedding immunocytochemistry was performed on ultrathin sections
obtained from two adult Wistar rats after intracardiac perfusion with
2% dextran (molecular weight 70,000) followed by the fixative (2.5%
glutaraldehyde + 1% paraformaldehyde in 0.1 M phosphate buffer, pH
7.4) under deep anesthesia (pentobarbital 50 mg/kg). Sections were
incubated with previously characterized primary antibodies raised in
rabbits against glutaraldehyde conjugates of glutamate or GABA (26,
27). Labeling was visualized by gold particles (mean diameter 15 nm),
coupled to the secondary antibody. The relationship between gold
particle density and the actual concentration of amino acid varied
depending on the antibody or experimental conditions (26, 27).
Specificity was assessed by using test sections containing
glutaraldehyde conjugates of selected amino acids and revealed no cross
reactivity of the antibodies except for a slight cross reactivity of
the glutamate antiserum with glutamine (not shown). To quantify
immunoreactivity associated with synaptic vesicles, locations of gold
particles, centers of vesicles, and outlines of the profiles were
digitized from electron micrographs by using a modified version of the
program microtrace as described (28, 29). Custom software
was used to determine the intercenter distances from each gold particle
to the nearest synaptic vesicle (30), sorting the distances into 20-nm
bins (chosen arbitrarily as a distance slightly smaller that the
theoretical resolution of the immunogold localization). The
distribution was compared with that of distances from random points to
synaptic vesicle centers (28, 29). The random points, 1,000 times the
number of gold particles, were spread over the nerve terminal cytoplasm
(areas occupied by mitochondria were excluded from the analysis). The
statistical significance of the difference between the observed and
expected (random points) distributions among three bins (0–20 nm,
20–40 nm, >40 nm) was evaluated by the χ2
test for known distributions.

Results

Glutamate released from individual MC dendrites is proposed to
have both local and diffuse actions resulting respectively in self- and
lateral excitation of MC via the activation of
N-methyl-d-aspartate (NMDA) receptors (20–22).
To locate and study more precisely these receptors, we began by
iontophoretically applying l-glutamate at the
somato-dendritic compartment of MC (Fig.
1). Rat olfactory bulb slices were bathed
in Mg2+-free buffer to remove the antagonistic
action of Mg2+ on NMDA channels, supplemented
with TTX (0.5 μM) to block Na+ action
potentials. Whole-cell patch-clamp recordings in the presence of the
non-NMDA receptor antagonist NBQX (10 μM) and BMI (10–20 μM) with
PTX (100 μM) and strychnine (30 μM), to block both GABA type A and
glycine receptors, revealed an inward current in response to glutamate
iontophoresis (Fig. 1A). Focal application of
glutamate to MC always elicited responses but small (≈20 μm)
displacements of the ejecting pipette from the soma and along the
lateral dendrite either increased or decreased the amplitude of the
responses (data not shown; n = 6). This observation
indicates an uneven distribution of functional glutamate receptors
activated by the iontophoresis. To compare iontophoretic responses with
synaptically released glutamate, MC were subjected to a brief
depolarization (from −70 to +10 mV, 25 ms), which evoked
endogenous glutamate release (refs. 7, 8, and 22; Fig. 1A and B). Under these conditions, the short
depolarizing step evoked after the iontophoresis application induced an
inward current interpreted as self-excitation produced by locally
released glutamate (19–22).

MC lateral dendrites express NMDA receptors with unusual pharmacology.
(A) Sequential iontophoresis of glutamate and voltage
steps were performed on a MC. Brief glutamate application (filled
arrowhead, 500 ms) elicited a direct inward current while a short
depolarization applied through the recording electrode (open arrowhead;
depolarizing currents were blanked) induced an evoked inward current.
Recordings were performed in control conditions (Ctrl: black trace; 0
Mg2+, 0.5 μM TTX, 10 μM BMI, 100 μM PTX, 10 μM
NBQX). Red trace indicates application of
d,l-APV (100 μM), and blue trace indicates
application of 5,7-dCK (15 μM). Each trace is the average of five
sweeps. (Inset) Infrared differential interference
contrast image of a MC showing a lateral dendrite (arrow), the axon
(*) and the location of the recording and ejecting pipettes.
(B) Time course of the experiment illustrated in
A. The colored boxes represent the duration of bath
applications of the antagonists. (C) Summary graph showing the
percentage of blockage induced by different NMDA receptor antagonists
on iontophoretic (filled columns) and voltage-evoked (open columns)
responses. Cells were recorded in the same external medium as in
A supplemented with different NMDA receptor antagonists.
The application of d,l-APV (100 μM) induced a
clear blockade of the evoked response whereas iontophoretic responses
were slightly potentiated (not statistically significant). Application
of 1.3 mM Mg2+ inhibited both responses (a mean reduction
of 70 ± 4%, P < 0.0001,
n = 10 and 83 ± 4%, P <
0.009, n = 6, for iontophoresis and evoked
responses, respectively). Application of 25 μM MK-801 also inhibited
both responses (a mean reduction of 86 ± 2%,
P < 0.0001, n = 12 and 86
± 5%, P < 0.04, n = 6, for,
respectively, iontophoresis and evoked responses). For iontophoresis,
the control medium was supplemented with
d,l-APV (100 μM). (D) A MC
exhibits spontaneous EPSCs (spont EPSCs) mediated in part by
APV-resistant NMDA receptors. MC recorded under control conditions: 0
Mg2+, 10 μM BMI, 100 μM PTX, 10 μM NBQX, and 100 μM
d,l-APV showed spontaneous EPSCs
(Top, the red trace represents the average of 50 events
whereas the other traces represent single events). Bath application of
5,7-dCK (15 μM) did not affect their frequency (see histogram; bin
10 s) but decreased their decay time constant
(Middle; the red trace represents the average of 70
events). (Bottom) The two averaged traces are scaled to
match peak amplitudes. The remaining component of the EPSC is mediated
by the non-NMDA receptors because they were totally blocked by NBQX (10
μM). (Scale bars: 2 ms and 10 pA; see Table 1, which is published as
supplemental data on the PNAS web site, www.pnas.org.)

APV Reveals an Unusual NMDA Receptor in MC.

We used specific antagonists of ionotropic glutamate receptors to
further characterize iontophoretic responses and self-excitation
induced by exogenous and endogenous glutamate,
respectively. The NMDA receptor antagonist
d,l-APV was first added to the external
recording medium already containing NBQX, PTX, and BMI (Fig. 1A–C). Surprisingly, whereas the application of
APV (100 μM) was able to nearly abolish the voltage pulse-evoked
responses as reported by others (86 ± 3% reduction,
n = 9; P < 0.02), iontophoretic
glutamate responses were either unaffected or even potentiated in the
presence of APV (17 ± 8% increase, n = 18;
P = 0.16; see Fig. 1A and B).
These results were partially mimicked by the use of a different
competitive NMDA receptor antagonist for the glutamate binding site
CPP. In all cells tested, bath-applied CPP (10 μM) strongly and
reversibly reduced the voltage pulse-evoked current (84 ± 3%
reduction; n = 5; P < 5 ×
10−5) whereas the iontophoresis-induced currents
were only slightly affected (14 ± 2% reduction,
n = 5; P < 0.02; Fig. 1C).
Therefore, our protocol revealed the presence of glutamate receptors
located on the somato-dendritic compartment of MC (whose activation
resulted in an APV-resistant current) that were clearly distinct from
those activated by voltage pulses (resulting in an APV-sensitive
current). The APV-resistant inward current seen with our iontophoresis
protocol was, however, mediated by activation of NMDA receptors because
bath application of a competitive NMDA receptor antagonist specific for
the glycine binding site, dCK (15 μM), dramatically reduced its
amplitude by 90.4 ± 1.4% (n = 14,
P < 2 × 10−7; Fig. 1A–C).

To further characterize the properties of the APV-resistant inward
current, we studied its voltage dependency (supplemental Fig. 6). In
the presence of external Mg2+ and APV, the
current induced by bath application of NMDA (100 μM) showed the
typical rectification characteristic of NMDA currents and was found to
be blocked by dCK application (data not shown; n = 4).
Furthermore, we used a panel of other blockers known to specifically
target different sites of the NMDA receptors. The results from
experiments designed to compare iontophoresis-induced and
voltage-evoked responses are summarized in Fig. 1C. The two
NMDA channel pore blockers, MK-801 (25 μM) and
Mg2+ (1.3 mM), strongly reduced both responses.
Similar iontophoretic applications of glutamate were made onto the soma
of GC (bathed with 0 Mg2+, TTX, BMI, PTX,
strychnine, and NBQX) to test the effectiveness of APV. In contrast to
MC, iontophoretic applications on GC elicited an NMDA current, which
was fully blocked by 50 μM d-APV (supplemental Fig. 6;
n = 4). This finding rules out the possibility that
APV, a competitive antagonist, was inefficient due to a massive
application of exogenous glutamate (supplemental Fig. 7).

We further tested the relative sensitivity of NMDA receptors to bath
application of APV in two different synapses. First, olfactory nerve
stimulations were applied to recruit glutamatergic synapses made
between olfactory nerve axons and apical dendrites of MC or tufted
cells. Second, stimulations in the EPL were used to evoke excitatory
dendrodendritic synaptic responses between the MC or tufted cell
lateral dendrites and the dendrites of GC. As reported by others (7, 8,
13, 25, 31–35), an APV-sensitive component was detected at both
synapses. Furthermore, in contrast to results obtained during
iontophoresis, when APV and NBQX were sequentially applied, a total
blockade was seen for the two synaptic responses, indicating absence of
APV-resistant NMDA current at these synapses (data not shown;
n = 7 and 4, respectively).

From these results, we conclude that iontophoretically applied
glutamate reveals the presence of an unusual type of NMDA receptor
located on the somato-dendritic compartment of MC. We then investigated
whether this APV-resistant NMDA component could participate in synaptic
transmission. For this purpose, MC were voltage-clamped at −70 mV, in
part to reduce the probability of spontaneous release of glutamate, and
recorded in Mg2+-free saline supplemented with
BMI (10–20 μM), PTX (100 μM), and d,l-APV
(100 μM). Under these conditions, spontaneous EPSCs reflecting the
activation of both non-NMDA and NMDA glutamate receptors were detected
(Fig. 1D; n = 7) even when the apical MC
dendrite was truncated. Application of dCK (15 μM) caused a marked
change in the kinetics of these spontaneous events, revealing that the
non-NMDA receptor-mediated component had faster decay kinetics
(3.8 ± 0.6 ms in APV vs. 1.4 ± 0.2 ms in APV + dCK;
P < 0.02; see also traces in Fig. 1D).
Subsequent addition of NBQX (10 μM) eliminated the remaining synaptic
activity (n = 5).

MC Are Excited by Interneurons from the GC Layer.

It is noteworthy that these results contrast with previous
findings on self-excitation, which report only an NMDA component
(19–22). However, they are supported by recordings of voltage
pulse-induced responses found to be sensitive to 10 μM NBQX (a mean
reduction of 13 ± 5%, P < 0.009,
n = 10; not shown; see also ref. 51). Because MC
dendrites of mammals do not make any synaptic contacts with other MC
(2), it has been proposed that extrasynaptic spillover of glutamate
released from these cells can evoke self-excitation and excitatory
transmission between neighboring MC (19–22). However, only an
APV-sensitive component was believed to mediate these effects. Thus,
the source of glutamate able to activate both non-NMDA and NMDA
receptors in the presence of APV must be different from the one
previously thought. To identify its origin, we performed
immunocytochemical studies to detect glutamate-containing spines within
the olfactory bulb network.

Ultrastructural immunogold cytochemistry revealed glutamate-like
immunoreactivity (Glu-LI) in dendritic terminals making reciprocal
synapses with MC dendrites in the EPL. Based on their morphological
features (2, 3), these terminals were identified as GC spines (Fig.
2A). As previously reported
for GC spines (36), the cross-sectioned area occupied by mitochondria
represented 4.1 ± 1.1% of the total area of the profiles
(n = 24). In another set of experiments, immunogold
detection of GABA was performed, and anatomically similar spines were
found to be GABA-like immunoreactive (GABA-LI) (Fig.
2B), in accordance with their inhibitory function
(7–10, 37, 38). In presynaptic profiles, glutamate may serve as GABA
precursor. Alternatively, it could be used as a transmitter that would
imply a vesicular localization of Glu-LI (30, 39, 40). This issue was
addressed by calculating intercenter distances between gold particles
and the nearest synaptic vesicle (28, 29). In GC spines labeled for
glutamate, short distances (less than 20 nm) are significantly more
frequent than expected from a random distribution of gold particles
over the spines, indicating that glutamate is associated with vesicles
(Fig. 3A). In GABA-LI-positive
GC spines, gold particles were similarly found to be significantly
associated with the synaptic vesicles (Fig. 3B). A positive
control for vesicular glutamate labeling was obtained from the
glutamatergic primary olfactory terminals (41), showing a nonrandom
intercenter distance distribution shifted toward short distances (Fig.
3C). By contrast, axon terminals from centrifugal neurons,
thought to be principally nonglutamatergic and to impinge on GC
dendrites within the EPL (3, 42), exhibit a randomly distributed Glu-LI
(Fig. 3D). Our data suggest that glutamate is preferentially
located in vesicles contained in presynaptic GC spines.

Quantification of the association of immunoreactivity with synaptic
vesicles. Measurements of intercenter distances between each gold
particle and the nearest synaptic vesicle were done, and distances were
sorted into bins of 20 nm (columns), the y axis showing
percent of total in each bin. Distances to the vesicle center from
points randomly distributed over the terminal (●)
also were calculated (see refs. 40 and 41). (A) Short
distances were significantly more represented in experimental
distributions compared with random distributions in GC spines labeled
for glutamate, indicating that Glu-LI was associated with synaptic
vesicles. (B) GABA-LI also was localized to vesicles in
GC (Gr) spines. (C) The same was true for Glu-LI in the
primary olfactory axon terminals (Tolf) within the glomeruli (GL).
(D) By contrast, axon terminals (Tax) from centrifugal
neurons making asymmetric contacts with GC dendrites in the EPL (3)
contained glutamate not associated with synaptic vesicles
(χ2 test). Gr Glutamate: 500 gold particles (24 spines);
Gr GABA: 260 gold particles (21 spines); Tolf glutamate: 387 gold
particles (13 terminals); Tax: 282 gold particles (11 terminals). Bins
not shown amounted to less than 2% of total. χ2 values
for comparison of gold particles distribution with random points
distribution: Gr (EPL) glutamate, 49.6; Gr (EPL) GABA, 87.6; Tolf GL
glutamate, 20.7; Tax EPL glutamate, 2.1.

Activation of GC Elicits Excitatory Synaptic Events in MC.

We then asked whether this vesicular glutamate could be released
by direct activation of GC. To address this question, MC were recorded
and GC were directly stimulated by using somatic glutamate application.
Fig. 4A illustrates a MC
recording during local stimulations of GC. In
Mg2+-free saline, granule cell stimulation
elicited large synaptic responses in MC (Fig. 4B1). Because
the recording pipette contained a Cs/gluconate-based internal
solution that sets the reversal potential for GABA type A
receptor-mediated currents near −50 mV, inhibitory postsynaptic
currents (IPSCs) were seen as inward events at a holding potential of
−70 mV. These evoked synaptic currents were reduced by adding PTX (100
μM) with BMI (10–20 μM), indicating they were mediated by MC GABA
type A receptors (Fig. 4B1). Focal stimulations of GC
trigger evoked synaptic responses in MC resulting from the summation of
many individual IPSCs (Fig. 4B2). In the cell displayed in
Fig. 4B3, the average IPSC (n = 90 events)
had a peak amplitude of −38 pA with a decay time constant of 9 ms.
Thus, inhibitory inputs onto MC can be reliably activated by glutamate
applied to GC soma (see Fig. 4B1). Interestingly, after the
blockade of GABA type A receptors, large spontaneously occurring
excitatory currents appeared on MC. As reported (20–22, 25), bath
application of d,l-APV (100
μM) abolished these events (Fig. 4A) but revealed
slow inward currents and discrete synaptic events that could still be
detected in the presence of APV and IPSCs antagonists (Fig. 4C1 and C2). As shown by the application of dCK,
these synaptic currents were excitatory (a mean amplitude of −10 pA)
and were mediated by activation of both non-NMDA and NMDA receptors
(Fig. 4C3). After addition of dCK, the averaged EPSC had a
fast decay time constant (3.6 ms) characteristic of non-NMDA
receptor-mediated EPSC (see Fig. 1D). Moreover, the slow
inward current was reversibly reduced either by 25 μM MK-801 or 15
μM dCK (n = 5; Fig. 4D). The possibility
that this slow inward response was due to glutamate spreading directly
onto MC was discarded on the following grounds. Given the separation
between iontophoresis and recording electrodes (at least 100 μm), its
seems unlikely that glutamate applied in the GC layer could diffuse so
far away. Second, bath application of TTX (0.5 μM) completely blocked
this evoked response (n = 4), demonstrating that
stimulation with iontophoresis of glutamate requires neuronal spiking
(Fig. 4D).

Stimulation of GC elicited glutamate release onto MC.
(A) MC are recorded while GC are stimulated by the
iontophoresis of glutamate. Traces represent the time course of the
experiment and were cut during application of BMI (10 μM) and PTX
(100 μM) because they produced large inward currents that were
blocked by d,l-APV (100 μM).
(B 1–3) GC stimulation increases GABA
release onto MC. (B1) Experiment performed in
0-Mg2+ during iontophoresis stimulations (black arrows).
The histogram represents the IPSC frequency during the recording (bin
1 s). BMI/PTX reduced those events. (B2) The
stimulation increases the IPSC frequency (Upper, see
also the histogram in B1; the step represents
iontophoretical applications). (Lower) Shown is an
expanded time scale part of the upper trace showing individual synaptic
events. (B3) Unitary IPSC (Upper) and the
average of 90 events (Lower) are presented. (C
1–3) GC stimulation increases glutamate release onto MC.
(C1) The same cell as in B was recorded
in: 0 Mg2+, 10 μM BMI, 100 μM PTX, and 100 μM
d,l-APV (black arrows indicate iontophoretic
stimulations). The histogram represents EPSC frequency during the
recording (bin 1 s). The bar shows the duration of dCK (15 μM)
application. (C2) Traces showing the effect of one
iontophoretic stimulation. Stimulation of GC induced a slow inward
current (Upper) on the top of which discrete EPSCs could
be seen (lower trace represents an expanded part of the upper one; see
also histogram in C1; the step represents the
iontophoretic application). (C3) Averages of EPSC
recorded with or without dCK (15 μM). (Upper) Average
of 35 events recorded before dCK application. (Lower)
Changes in the decay time after dCK application (the average in dCK
represents the average of 20 events). (D)
(Left) Graph represents the amplitude of the
iontophoretic evoked inward current recorded during the time course of
the experiment depicted in C1. Bath application of dCK
(15 μM) revealed that this current was mediated in part by
APV-resistant NMDA receptors. After washout of this drug, bath
application of TTX (0.5 μM) completely abolished the current,
confirming that it was due to the excitation of GC rather than a direct
effect on the MC dendrites. The traces showing the drug effects are the
average of five sweeps.

We next took advantage of the unique way in which the transient A-type
potassium current (IA) specifically regulates
neurotransmitter release from GC, to unambiguously locate the source of
glutamate activating ionotropic receptors on MC. Recently, the
transient IA has been demonstrated to prevent
GABA release mediated by non-NMDA receptors (13). We investigated
whether IA also could be responsible for limiting
feedback excitation. Depolarizing the recorded MC (25 ms from −70 to +
10 mV) in 1.3 mM Mg2+ saline containing BMI- and
PTX-induced reciprocal EPSCs. As expected if glutamate originates from
GC, addition of a specific antagonist of IA,
4-aminopyridine (4-AP; 2.5–5 mM) markedly increased the amplitude of
the reciprocal excitation (76 ± 13% increase, n
= 5; P < 0.015; Fig.
5A). Similarly, we have tested
whether glutamate released by GC also could be involved in lateral
excitation between MC through synaptically shared GC. Electrical
stimulations were delivered at the border between the glomerular layer
and EPL (see Fig. 5B for the experimental configuration) to
activate neighboring apical MC dendrites. To avoid direct stimulation
of the recorded MC (and therefore reciprocal excitation), the sodium
channel antagonist QX-314 (10 mM) was added to the patch pipette
solution, and MC were always recorded at a certain distance from the
stimulating electrodes (at least 200 μm). Under these conditions, the
amplitude of lateral excitation was similarly potentiated by the
blockade of IA (86 ± 35% increase,
n = 5; P < 0.02; Fig. 5B).

Glutamate released by GC mediates both reciprocal and lateral
excitation. (A) The reciprocal excitation elicited by
depolarization of the recorded MC (Left) is recorded in
control conditions (1.3 mM Mg2+ with 10 μM BMI and 100
μM PTX). 4-AP (5 mM) increased significantly the amplitude response
(middle traces) in five tested cells. (Right) Summary
graph of the 4-AP effect. (B) EPL stimulation triggered
lateral excitation recorded in control conditions containing
Mg2+ (1.3 mM) with BMI (10 μM), PTX (100 μM), and
QX-314 (10 mM) in the recording pipette. 4-AP (5 mM) also increased
significantly the amplitude response (middle traces) in
five tested cells. (Right) Summary graph of the 4-AP
effect. (C) Model representing the two reciprocal
synapses mediated by the same or different subtypes of GC. The term
reciprocal implies that MC releases glutamate, activating both non-NMDA
and APV-sensitive NMDA receptors, which trigger neurotransmitter
release by GC spines at the same synapse (arrows). Glutamate from GC
activates both non-NMDA and APV-resistant NMDA receptors on the MC
dendrites. It is possible that glutamate released by MC could activate
directly the MC glutamate receptors (dotted arrow) although our data
indicate a moderate contribution (supplemental Fig. 8).

Discussion

Synaptic transmission at dendrodendritic synapses between MC and
GC provides a fast inhibitory feedback onto MC. Our results showed that
GC also contribute to recurrent excitation of relay neurons. These
interneurons release glutamate, which activates both AMPA and
APV-insensitive NMDA receptors located on the soma and lateral
dendrites of MC. This recurrent excitation onto relay neurons is likely
to play an important role in prolonging periods of phasic firing in MC.

MC Lateral Dendrites Receive Excitatory Inputs from GC.

The idea that odor information is encoded by activity distributed
across the olfactory bulb neuronal network is consistent with
physiological experiments (43–45), but the underlying mechanism
remains poorly documented. Through their tangential extent and numerous
dendrodendritic reciprocal synapses with granule cell dendrites (2, 4,
5), MC lateral dendrites provide a key anatomical element for
distributing information throughout the entire olfactory bulb. The
temporal and spatial shaping of olfactory bulb output is strongly
influenced by the amplitude and duration of the dendrodendritic
inhibition (44, 45). However, theoretical studies have suggested that
excitation also may play an important role in temporal shaping of
neuronal discharges (46). Experimentally, excitatory events in MC can
be induced by antidromic stimulations (19, 25) or intracellular
depolarizing currents (20–22, 51), demonstrating the presence of
recurrent excitatory inputs to MC mediated by NMDA receptors.
Preembedding immunocytochemistry has demonstrated ionotropic glutamate
receptors in MC dendrites (47–49), but a recent postembedding
immunogold analysis revealed no labeling for NMDA receptors and only
occasional labeling for AMPA receptors on MC dendrite membranes facing
GC spines (50). The lack of immunodetection of NMDA receptors could be
related to the pharmacological profile reported here. Alternatively,
these receptors may be scattered rather than clustered, preventing easy
detection by immunogold labeling. A similar mechanism may explain why
non-NMDA receptors that contribute to MC excitation have escaped
immunogold detection.

The major synaptic contacts of secondary MC dendrites within the EPL
are reciprocal synapses with GC spines (1, 37, 38). However, a small
subpopulation of interneurons distinct from GC and expressing
parvalbumin also makes reciprocal contacts with MC dendrites in the EPL
(36). These terminals were found to be rich in mitochondria and thus
can be distinguished from GC spines. Furthermore, parvalbumin-positive
cell bodies and processes are confined to the EPL whereas
glutamate-mediated events in MC were recorded after stimulations
applied in the GC layer. On these grounds, the large majority of the
cellular profiles, detected by Glu-LI and participating in reciprocal
synapses with MC, clearly belongs to the GC population. We show here
that dendritically released glutamate from MC activates local
interneurons in the GC layer, through NMDA (APV-sensitive) receptors.
Then, GC subsequently excite MC by releasing glutamate on both non-NMDA
receptors and an APV-resistant subtype of NMDA receptor (see model in
Fig. 5C). Because feedback excitation is almost completely
blocked by APV, it would appear that the APV-resistant receptors are
not reached by glutamate of MC origin.

An Excitatory Synaptic Connection in the Main Olfactory Bulb.

Recent studies have proposed that recurrent excitation of MC is
mediated by glutamate that escapes from synapses to activate
exclusively extrasynaptic NMDA receptors. However, it is questionable
whether this spillover-mediated transmission plays a functionally
important role in synaptic communication because previous studies were
performed in the absence of external Mg2+.
Furthermore, the presence of non-NMDA synaptic events in MC lateral
dendrites reported here is in apparent contrast with the spillover
hypothesis because activation of non-NMDA receptors requires high
concentration of glutamate, probably achieved only within the synaptic
cleft. Thus, non-NMDA synaptic events indicate that transmission at the
excitatory dendrodendritic synapses is rather “point to point,”
with glutamate being released from both MC and GC. Because GC vastly
outnumber MC, the activation of a single MC could result in robust
reciprocal dendrodendritic excitation. Whether the same or different GC
release both glutamate and GABA is currently unknown and should be
specifically addressed in the near future.

In conclusion, we have demonstrated that the main population of bulbar
local interneurons, previously thought to be exclusively inhibitory,
provides both strong feedback and lateral excitation on MC through a
dendrodendritic excitatory reciprocal synapse. This finding points to
an unexpected degree of complexity of the neuronal network of the main
olfactory bulb and forces us to revise current concepts about the way
the GC regulate the olfactory bulb output.

Acknowledgments

We thank B. Riber and K. M. Gujord for excellent technical
assistance and F. Jourdan and H. McLean for comments on the manuscript.
This work was supported in part by Centre National de Recherche
Scientifique (A.D. and P.-M.L.), a grant from the French
Ministère de la Recherche et de l'Enseignement (A.C.) and the
Ministère de l'Education Nationale, de la Recherche et de la
Technologie (ACI Biologie du Développement et Physiologie
Intégrative 2000) (to P.-M.L.), a fellowship under the
Organization for Economic Cooperation and Development Cooperative
Research Program and Biological Resource Management for Sustainable
Agricultural Systems (A.D.), and the Norwegian Research Council
(J.G.B., O.P.O., and J.S.-M.).

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